Ezh2 inhibitors for treating lymphoma

ABSTRACT

The present invention relates to compositions comprising inhibitors of human histone methyltransferase EZH2 and their use for the treatment of cancer.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/454,835, filed Jun. 27, 2019, which is a continuation of U.S. patentapplication Ser. No. 16/181,747, filed Nov. 6, 2018 (now abandoned),which is a continuation of U.S. patent application Ser. No. 15/319,535,filed Dec. 16, 2016 (now U.S. Pat. No. 10,166,238), which is a U.S.National Phase Application, filed under 35 U.S.C. § 371, ofInternational Application No. PCT/US2015/036310, filed Jun. 17, 2015,which claims the benefit of and priority to U.S. Patent Application Nos.62/013,522, filed Jun. 17, 2014; and 62/036,265, filed Aug. 12, 2014.The contents of each of these applications are hereby incorporated byreference in their entireties.

BACKGROUND OF THE INVENTION

EZH2, a histone methyltransferase, has been associated with variouskinds of cancers. Specifically, mutations and and/or overactivity ofEZH2 are found in a range of cancers, such as lymphomas, leukemias andbreast cancer. There is an ongoing need for new agents as EZH2inhibitors for use in anticancer treatment.

SUMMARY OF THE INVENTION

In one aspect, the present disclosure features a method for thetreatment or prevention of non-Hodgkin's lymphoma (NHL) in a subject inneed thereof. The method includes administration of a therapeuticallyeffective amount of an EZH2 inhibitor to said subject.

The method can include one or more of the following features.

In one embodiment, the NHL is selected from diffuse large B-celllymphoma (DLBCL), a germinal center-derived lymphoma, a non-germinalcenter-derived lymphoma, follicular lymphoma (FL), primary mediastinallarge B-cell lymphoma (PMBCL), marginal zone lymphoma (MZL), Burkitt'slymphoma and other non-Hodgkin's lymphoma subtype.

In one embodiment, the NHL is a germinal center-derived lymphoma.

In one embodiment, the NHL is a non-germinal center-derived lymphoma.

In one embodiment, the NHL is follicular lymphoma.

In one embodiment, the NHL is PMBCL.

In one embodiment, the NHL is marginal zone lymphoma.

In one embodiment, the NHL is Burkitt's lymphoma.

In one embodiment, the NHL is other Non-Hodgkin's lymphoma subtype.

In one embodiment, the NHL is an EZH2 wild type B-cell lymphoma, e.g.,the NHL cells having non-mutated, wild-type EZH2 protein.

In one embodiment, the NHL is an EZH2 mutant B-cell lymphoma, e.g., theNHL cells having mutant EZH2 protein.

In certain embodiments, the non-germinal center-derived lymphomas isActivated B-Cell (ABC) lymphoma.

In one embodiment, the non-germinal center B-cell lymphoma is an EZH2wild type B-cell lymphoma, e.g., the lymphoma cells having non-mutated,wild-type EZH2 protein.

In another embodiment, the non-germinal center B-cell lymphoma is anEZH2 mutant B-cell lymphoma, e.g., the lymphoma cells having mutant EZH2protein.

In one embodiment, the germinal center-derived lymphoma is an EZH2 wildtype B-cell lymphoma, e.g., the lymphoma cells having non-mutated,wild-type EZH2 protein.

In another embodiment, the germinal center-derived lymphoma is an EZH2mutant B-cell lymphoma, e.g., the lymphoma cells having mutant EZH2protein.

In one embodiment, the follicular lymphoma (FL), primary mediastinallarge B-cell lymphoma (PMBCL), or marginal zone lymphoma (MZL) is anEZH2 wild type germinal center B-cell lymphoma, e.g., the germinalcenter B-cell lymphoma cells having non-mutated, wild-type EZH2 protein.

In another embodiment, the follicular lymphoma (FL), primary mediastinallarge B-cell lymphoma (PMBCL), or marginal zone lymphoma (MZL) is anEZH2 mutant germinal center B-cell lymphoma, e.g., the germinal centerB-cell lymphoma cells having mutant EZH2 protein.

In one embodiment, the EZH2 inhibitor is administered orally.

In one embodiment, the subject is a human being.

In one embodiment, the EZH2 inhibitor is EPZ-6438 (tazemetostat), havingthe following formula:

or a pharmaceutically acceptable salt thereof

In one embodiment, the EZH2 inhibitor is administered to the subject ata dose of approximately 100 mg to approximately 3200 mg daily.

In one embodiment, the EZH2 inhibitor is administered to the subject ata dose of approximately 100 mg BID to approximately 1600mg BID.

In one embodiment, the EZH2 inhibitor is administered to the subject ata dose of approximately 100 mg BID, 200 mg BID, 400 mg BID, 800 mg BID,or 1600 mg BID.

In one embodiment, the EZH2 inhibitor is either compound (A), (B), (C),or (D):

or pharmaceutically acceptable salts or solvates thereof.

Any of the above aspects and embodiments can be combined with any otheraspect or embodiment.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. In the specification, thesingular forms also include the plural unless the context clearlydictates otherwise. Although methods and materials similar or equivalentto those described herein can be used in the practice or testing of thepresent invention, suitable methods and materials are described below.All publications, patent applications, patents and other referencesmentioned herein are incorporated by reference. The references citedherein are not admitted to be prior art to the claimed invention. In thecase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods and examples areillustrative only and are not intended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and further features will be more clearly appreciated from thefollowing detailed description when taken in conjunction with theaccompanying drawings.

FIG. 1 shows the summary of first-in-human phase 1 trial withtazemetostat (EPZ-6438) E7438-G000-001 (NCT01897571).

FIG. 2 shows the patient tumor types.

FIG. 3 shows the NHL patient demographics.

FIG. 4 shows the pharmacokinetics of tazemetostat (EPZ-6438).

FIG. 5 shows the PK-PD: EZH2 inhibition in surrogate tissue.

FIG. 6 shows the adverse events of the trial.

FIG. 7 shows the overall best responses in NHL patients.

FIG. 8 shows the target lesion activity.

FIG. 9 shows the CR (complete response) in primary mediastinal B-celllymphoma.

FIG. 10 shows the CR in follicular lymphoma with wild type EZH2.

FIG. 11 shows the response in DLBCL with mutant EZH2 lymphoma (Y646H).

DETAILED DESCRIPTION OF THE INVENTION

EZH2 is a histone methyltransferase that is the catalytic subunit of thePRC2 complex which catalyzes the mono- through tri-methylation of lysine27 on histone H3 (H3-K27). Histone H3-K27 trimethylation is a mechanismfor suppressing transcription of specific genes that are proximal to thesite of histone modification. This trimethylation is known to be acancer marker with altered expression in cancer, such as prostate cancer(see, e.g., U.S. Patent Application Publication No. 2003/0175736;incorporated herein by reference in its entirety). Other studiesprovided evidence for a functional link between dysregulated EZH2expression, transcriptional repression, and neoplastic transformation.Varambally et al. (2002) Nature 419(6907):624-9 Kleer et al. (2003) ProcNatl Acad Sci USA 100(20):11606-11.

EZH2 methylation activity plays an important role in the regulation andactivation of germinal center B-cells. EZH2 protein levels increasefollowing the activation of B-cells. Following activation, B-cells takeresidence in the germinal center of lymphoid organs, wherein somatichypermutation occurs, a process associated with the repression ofanti-apoptotic genes and check point regulators. EZH2 methylating eventstarget genes that are involved in B-cell proliferation, differentiationand maturation, including CDKN1A (role in cellular proliferation), PRDM1(role in B-cell differentiation) and IRF4 (role in B-celldifferentiation).

Following the maturation and exit of B-cells from the germinal center,there is a reduction of the levels of EZH2 within the B-cells. However,EZH2 presence and activity after B-cell maturation is associated withseveral kinds of lymphomas including germinal center B-cell lymphoma,among others.

Aberrant activation or misregulation of EZH2 is found in many commonsubtypes of non-Hodgkin lymphoma (NHL): diffuse large B cell lymphoma(DLBCL), germinal center B-cell like diffuse large B-cell lymphoma (GCBDLBCL), non-germinal center B-cell like diffuse large B-cell lymphomaincluding activated-B cell lymphoma (ABC DLBCL), Burkitt's lymphoma andother subtypes of non-Hodgkin lymphoma. Aberrant activation of ormisregulation EZH2 is also found in follicular lymphoma (FL), PrimaryMediastinal Large B-Cell Lymphoma (PMBCL) and marginal zone lymphoma(MZL).

Genetic alterations within the EZH2 gene are associated with alteredhistone methylation patterns. EZH2 mutations leading to the conversionof amino acid Y641 (equivalent to Y646, catalytic domain), to either F,N, H, S or C results in hypertrimethylation of H3K27 and driveslymphomagenesis. Additional genetic alterations that affect themethylation of H3K27 include EZH2 SET-domain mutations, overexpressionof EZH2, overexpression of other PRC2 subunits, loss of functionmutations of histone acetyl transferases (HATs), and loss of function ofMLL2. Cells that are heterozygous for EZH2 Y646 mutations result inhypertrimethylation of H3K27 relative to cells that are homozygouswild-type (WT) for the EZH2 protein, or to cells that are homozygous forthe Y646 mutation.

An aspect of the present invention relates to a method for treating oralleviating a symptom of a NHL in a subject in need thereof byadministering to the subject a therapeutically effective amount of anEZH2 inhibitor. An aspect of the present invention relates to a methodfor treating or alleviating a symptom of NHL in a subject in needthereof by administering to the subject a therapeutically effectiveamount of an EZH2 inhibitor. Another aspect of the present inventionrelates to a method for treating or alleviating a symptom of GCB DLBCLin a subject in need thereof by administering to the subject atherapeutically effective amount of an EZH2 inhibitor. In yet anotheraspect the present invention relates to a method for treating oralleviating a symptom of a FL, PMBCL, or MZL in a subject in needthereof by administering to the subject a therapeutically effectiveamount of an EZH2 inhibitor. The subject suitable for the method oftreatment described herein can either express a mutant EZH2 or awild-type EZH2 or has a mutation in the EZH2 gene or has a wild-typeEZH2 gene.

As described herein, inhibition of EZH2 activity significantly abrogatesthe division of the malignant cells.

In one embodiment, the EZH2 inhibitor is administered orally.

In one embodiment, the subject is a human being.

In any of the above aspects or embodiments, the invention also relatesto methods for detecting levels of histone methylation, e.g., H3K27trimethylation, in a skin biopsy. Histone methylation is detected priorto initiation of treatment, while the subject is receiving treatment,and/or after treatment has concluded.

The mutant EZH2 described herein refers to a mutant EZH2 polypeptide ora nucleic acid sequence encoding a mutant EZH2 polypeptide. In certainembodiments the mutant EZH2 comprises one or more mutations in itssubstrate pocket domain. For example, the mutation may be asubstitution, a point mutation, a nonsense mutation, a missensemutation, a deletion, or an insertion. Methods for detecting EZH2mutations have been described in PCT/US11/051258, PCT/US13/030565,US20150099747, each of which is incorporated herein by reference in itsentirety.

For purposes of this application, a Y641 mutant of human EZH2, and,equivalently, a Y641 mutant of EZH2, is to be understood to refer to ahuman EZH2 in which the amino acid residue corresponding to Y641 ofwild-type human EZH2 is substituted by an amino acid residue other thantyrosine.

Compounds suitable for the methods disclosed herein are described inU.S. Publications 20120264734, and 20140107122 the contents of which arehereby incorporated by reference in their entireties. The compounds ofsuitable for administration as part of a combination therapy with one ormore other therapeutic agents or treatment modality, suitable to beadministered together, sequentially, or in alternation.

In one embodiment, the compound suitable for the methods disclosedherein is EPZ-6438 (tazemetostat):

or a pharmaceutically acceptable salt thereof.

EPZ-6438 or a pharmaceutically acceptable salt thereof, as describedherein, is potent in targeting both WT and mutant EZH2. EPZ-6438 isorally bioavailable and has high selectivity to EZH2 compared with otherhistone methyltransferases (i.e. >20,000 fold selectivity by Ki).Importantly, EPZ-6438 has target methyl mark inhibition that results inthe killing of genetically defined cancer cells in vitro. Animal modelshave also shown sustained in vivo efficacy following inhibition oftarget methyl mark. Clinical trial results described herein alsodemonstrate the safety and efficacy of EPZ-6438.

In one embodiment, EPZ-6438 or a pharmaceutically acceptable saltthereof is administered to the subject at a dose of approximately 100 mgto approximately 3200 mg daily, such as about 100 mg BID to about 1600mgBID (e.g., 100 mg BID, 200 mg BID, 400 mg BID, 800 mg BID, or 1600 mgBID), for treating a NHL. On one embodiment the dose is 800 mg BID.

In some embodiments, a compound (e.g., EZH2 inhibitor) that can be usedin any methods presented here is:

or stereoisomers thereof or pharmaceutically acceptable salts andsolvates thereof.

In certain embodiments, a compound that can be used in any methodspresented here is Compound E:

or pharmaceutically acceptable salts thereof.

In some embodiments, a compound (e.g., EZH2 inhibitor) that can be usedin any methods presented here is GSK-126 having the following formula:

stereoisomers thereof, or pharmaceutically acceptable salts or solvatesthereof.

In certain embodiments, a compound that can be used in any methodspresented here is Compound F:

or stereoisomers thereof or pharmaceutically acceptable salts andsolvates thereof.

In certain embodiments, a compound (e.g., EZH2 inhibitor) that can beused in any methods presented here is any of Compounds Ga-Gc:

or a stereoisomer, pharmaceutically acceptable salt or solvate thereof.

In certain embodiments, a compound (e.g., EZH2 inhibitor) that can beused in any methods presented here is CPI-1205 or GSK343.

In one embodiment, the compound disclosed herein is the compound itself,i.e., the free base or “naked” molecule. In another embodiment, thecompound is a salt thereof, e.g., a mono-HCl or tri-HCl salt, mono-HBror tri-HBr salt of the naked molecule.

Compounds disclosed herein that contain nitrogens can be converted toN-oxides by treatment with an oxidizing agent (e.g.,3-chloroperoxybenzoic acid (mCPBA) and/or hydrogen peroxides) to affordother compounds suitable for any methods disclosed herein. Thus, allshown and claimed nitrogen-containing compounds are considered, whenallowed by valency and structure, to include both the compound as shownand its N-oxide derivative (which can be designated as N→O or N⁺O⁻).Furthermore, in other instances, the nitrogens in the compoundsdisclosed herein can be converted to N-hydroxy or N-alkoxy compounds.For example, N-hydroxy compounds can be prepared by oxidation of theparent amine by an oxidizing agent such as m-CPBA. All shown and claimednitrogen-containing compounds are also considered, when allowed byvalency and structure, to cover both the compound as shown and itsN-hydroxy (i.e., N—OH) and N-alkoxy (i.e., N—OR, wherein R issubstituted or unsubstituted C₁-C₆ alkyl, C₁-C₆ alkenyl, C₁-C₆ alkynyl,3-14-membered carbocycle or 3-14-membered heterocycle) derivatives.

“Isomerism” means compounds that have identical molecular formulae butdiffer in the sequence of bonding of their atoms or in the arrangementof their atoms in space. Isomers that differ in the arrangement of theiratoms in space are termed “stereoisomers.” Stereoisomers that are notmirror images of one another are termed “diastereoisomers,” andstereoisomers that are non-superimposable mirror images of each otherare termed “enantiomers” or sometimes optical isomers. A mixturecontaining equal amounts of individual enantiomeric forms of oppositechirality is termed a “racemic mixture.”

A carbon atom bonded to four nonidentical substituents is termed a“chiral center.”

“Chiral isomer” means a compound with at least one chiral center.Compounds with more than one chiral center may exist either as anindividual diastereomer or as a mixture of diastereomers, termed“diastereomeric mixture.” When one chiral center is present, astereoisomer may be characterized by the absolute configuration (R or S)of that chiral center. Absolute configuration refers to the arrangementin space of the substituents attached to the chiral center. Thesubstituents attached to the chiral center under consideration areranked in accordance with the Sequence Rule of Cahn, Ingold and Prelog.(Cahn et al., Angew. Chem. Inter. Edit. 1966, 5, 385; errata 511; Cahnet al., Angew. Chem. 1966, 78, 413; Cahn and Ingold, J. Chem. Soc. 1951(London), 612; Cahn et al., Experientia 1956, 12, 81; Cahn, J. Chem.Educ. 1964, 41, 116).

“Geometric isomer” means the diastereomers that owe their existence tohindered rotation about double bonds or a cycloalkyl linker (e.g.,1,3-cylcobutyl). These configurations are differentiated in their namesby the prefixes cis and trans, or Z and E, which indicate that thegroups are on the same or opposite side of the double bond in themolecule according to the Cahn-Ingold-Prelog rules.

It is to be understood that the compounds disclosed herein may bedepicted as different chiral isomers or geometric isomers. It shouldalso be understood that when compounds have chiral isomeric or geometricisomeric forms, all isomeric forms are intended to be included in thescope of the disclosure, and the naming of the compounds does notexclude any isomeric forms.

Furthermore, the structures and other compounds discussed in thisinvention include all atropic isomers thereof “Atropic isomers” are atype of stereoisomer in which the atoms of two isomers are arrangeddifferently in space. Atropic isomers owe their existence to arestricted rotation caused by hindrance of rotation of large groupsabout a central bond. Such atropic isomers typically exist as a mixture,however as a result of recent advances in chromatography techniques, ithas been possible to separate mixtures of two atropic isomers in selectcases.

“Tautomer” is one of two or more structural isomers that exist inequilibrium and is readily converted from one isomeric form to another.This conversion results in the formal migration of a hydrogen atomaccompanied by a switch of adjacent conjugated double bonds. Tautomersexist as a mixture of a tautomeric set in solution. In solutions wheretautomerization is possible, a chemical equilibrium of the tautomerswill be reached. The exact ratio of the tautomers depends on severalfactors, including temperature, solvent and pH. The concept of tautomersthat are interconvertable by tautomerizations is called tautomerism.

Of the various types of tautomerism that are possible, two are commonlyobserved. In keto-enol tautomerism a simultaneous shift of electrons anda hydrogen atom occurs. Ring-chain tautomerism arises as a result of thealdehyde group (—CHO) in a sugar chain molecule reacting with one of thehydroxy groups (—OH) in the same molecule to give it a cyclic(ring-shaped) form as exhibited by glucose.

Common tautomeric pairs are: ketone-enol, amide-nitrile, lactam-lactim,amide-imidic acid tautomerism in heterocyclic rings (e.g., innucleobases such as guanine, thymine and cytosine), imine-enamine andenamine-enamine. An example of keto-enol equilibria is betweenpyridin-2(1H)-ones and the corresponding pyridin-2-ols, as shown below.

It is to be understood that the compounds disclosed herein may bedepicted as different tautomers. It should also be understood that whencompounds have tautomeric forms, all tautomeric forms are intended to beincluded in the scope of the disclosure, and the naming of the compoundsdoes not exclude any tautomer form.

The compounds disclosed herein include the compounds themselves, as wellas their salts and their solvates, if applicable. A salt, for example,can be formed between an anion and a positively charged group (e.g.,amino) on an aryl- or heteroaryl-substituted benzene compound. Suitableanions include chloride, bromide, iodide, sulfate, bisulfate, sulfamate,nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate,glutamate, glucuronate, glutarate, malate, maleate, succinate, fumarate,tartrate, tosylate, salicylate, lactate, naphthalenesulfonate, andacetate (e.g., trifluoroacetate). The term “pharmaceutically acceptableanion” refers to an anion suitable for forming a pharmaceuticallyacceptable salt. Likewise, a salt can also be formed between a cationand a negatively charged group (e.g., carboxylate) on an aryl- orheteroaryl-substituted benzene compound. Suitable cations include sodiumion, potassium ion, magnesium ion, calcium ion, and an ammonium cationsuch as tetramethylammonium ion. The aryl- or heteroaryl-substitutedbenzene compounds also include those salts containing quaternarynitrogen atoms. In the salt form, it is understood that the ratio of thecompound to the cation or anion of the salt can be 1:1, or any rationother than 1:1, e.g., 3:1, 2:1, 1:2, or 1:3.

Additionally, the compounds disclosed herein, for example, the salts ofthe compounds, can exist in either hydrated or unhydrated (theanhydrous) form or as solvates with other solvent molecules. Nonlimitingexamples of hydrates include monohydrates, dihydrates, etc. Nonlimitingexamples of solvates include ethanol solvates, acetone solvates, etc.

“Solvate” means solvent addition forms that contain eitherstoichiometric or non stoichiometric amounts of solvent. Some compoundshave a tendency to trap a fixed molar ratio of solvent molecules in thecrystalline solid state, thus forming a solvate. If the solvent is waterthe solvate formed is a hydrate; and if the solvent is alcohol, thesolvate formed is an alcoholate. Hydrates are formed by the combinationof one or more molecules of water with one molecule of the substance inwhich the water retains its molecular state as H₂O.

As used herein, the term “analog” refers to a chemical compound that isstructurally similar to another but differs slightly in composition (asin the replacement of one atom by an atom of a different element or inthe presence of a particular functional group, or the replacement of onefunctional group by another functional group). Thus, an analog is acompound that is similar or comparable in function and appearance, butnot in structure or origin to the reference compound.

As defined herein, the term “derivative” refers to compounds that have acommon core structure, and are substituted with various groups asdescribed herein. For example, all of the compounds represented byFormula (I) are aryl- or heteroaryl-substituted benzene compounds, andhave Formula (I) as a common core.

The term “bioisostere” refers to a compound resulting from the exchangeof an atom or of a group of atoms with another, broadly similar, atom orgroup of atoms. The objective of a bioisosteric replacement is to createa new compound with similar biological properties to the parentcompound. The bioisosteric replacement may be physicochemically ortopologically based. Examples of carboxylic acid bioisosteres include,but are not limited to, acyl sulfonimides, tetrazoles, sulfonates andphosphonates. See, e.g., Patani and LaVoie, Chem. Rev. 96, 3147-3176,1996.

The present invention is intended to include all isotopes of atomsoccurring in the present compounds. Isotopes include those atoms havingthe same atomic number but different mass numbers. By way of generalexample and without limitation, isotopes of hydrogen include tritium anddeuterium, and isotopes of carbon include C-13 and C-14.

In certain aspects, “combination therapy” also embraces theadministration of the therapeutic agents as described above in furthercombination with other biologically active ingredients and non-drugtherapies (e.g., surgery or radiation treatment). Where the combinationtherapy further comprises a non-drug treatment, the non-drug treatmentmay be conducted at any suitable time so long as a beneficial effectfrom the co-action of the combination of the therapeutic agents andnon-drug treatment is achieved. For example, in appropriate cases, thebeneficial effect is still achieved when the non-drug treatment istemporally removed from the administration of the therapeutic agents,perhaps by days or even weeks.

In another aspect, a composition disclosed herein, or a pharmaceuticallyacceptable salt, solvate, analog or derivative thereof, may beadministered in combination with radiation therapy. Radiation therapycan also be administered in combination with a composition disclosedherein and another chemotherapeutic agent described herein as part of amultiple agent therapy.

A “pharmaceutical composition” is a formulation containing a compound ina form suitable for administration to a subject. A compound disclosedherein and one or more other therapeutic agents described herein eachcan be formulated individually or in multiple pharmaceuticalcompositions in any combinations of the active ingredients. Accordingly,one or more administration routes can be properly elected based on thedosage form of each pharmaceutical composition. Alternatively, acompound disclosed herein and one or more other therapeutic agentsdescribed herein can be formulated as one pharmaceutical composition.

In one embodiment, the pharmaceutical composition is in bulk or in unitdosage form. The unit dosage form is any of a variety of forms,including, for example, a capsule, an IV bag, a tablet, a single pump onan aerosol inhaler or a vial. The quantity of active ingredient (e.g., aformulation of the disclosed compound or salt, hydrate, solvate orisomer thereof) in a unit dose of composition is an effective amount andis varied according to the particular treatment involved. One skilled inthe art will appreciate that it is sometimes necessary to make routinevariations to the dosage depending on the age and condition of thepatient. The dosage will also depend on the route of administration. Avariety of routes are contemplated, including oral, pulmonary, rectal,parenteral, transdermal, subcutaneous, intravenous, intramuscular,intraperitoneal, inhalational, buccal, sublingual, intrapleural,intrathecal, intranasal, and the like. Dosage forms for the topical ortransdermal administration of a compound of this invention includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches and inhalants. In one embodiment, the active compound is mixedunder sterile conditions with a pharmaceutically acceptable carrier, andwith any preservatives, buffers, or propellants that are required.

As used herein, the phrase “pharmaceutically acceptable” refers to thosecompounds, anions, cations, materials, compositions, carriers, and/ordosage forms which are, within the scope of sound medical judgment,suitable for use in contact with the tissues of human beings and animalswithout excessive toxicity, irritation, allergic response, or otherproblem or complication, commensurate with a reasonable benefit/riskratio.

“Pharmaceutically acceptable excipient” means an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic and neither biologically nor otherwise undesirable, andincludes excipient that is acceptable for veterinary use as well ashuman pharmaceutical use. A “pharmaceutically acceptable excipient” asused in the specification and claims includes both one and more than onesuch excipient.

A pharmaceutical composition disclosed herein is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical), andtransmucosal administration. Solutions or suspensions used forparenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerin, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates, and agents for theadjustment of tonicity such as sodium chloride or dextrose. The pH canbe adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

A composition disclosed herein can be administered to a subject in manyof the well-known methods currently used for chemotherapeutic treatment.For example, for treatment of cancers, a compound disclosed herein maybe injected directly into tumors, injected into the blood stream or bodycavities or taken orally or applied through the skin with patches. Thedose chosen should be sufficient to constitute effective treatment butnot so high as to cause unacceptable side effects. The state of thedisease condition (e.g., cancer, precancer, and the like) and the healthof the patient should preferably be closely monitored during and for areasonable period after treatment.

The term “therapeutically effective amount”, as used herein, refers toan amount of a pharmaceutical agent to treat, ameliorate, or prevent anidentified disease or condition, or to exhibit a detectable therapeuticor inhibitory effect. The effect can be detected by any assay methodknown in the art. The precise effective amount for a subject will dependupon the subject's body weight, size, and health; the nature and extentof the condition; and the therapeutic or combination of therapeuticsselected for administration. For example, the therapeutically effectiveamount of an EZH2 inhibitor can be different for a patient having anEZH2 wild type germinal center B-cell lymphoma than for a patient havingan EZH2 mutant germinal center B-cell lymphoma. Therapeuticallyeffective amounts for a given situation can be determined by routineexperimentation that is within the skill and judgment of the clinician.

In certain embodiments the therapeutically effective amount of eachpharmaceutical agent used in combination will be lower when used incombination in comparison to monotherapy with each agent alone. Suchlower therapeutically effective amount could afford for lower toxicityof the therapeutic regimen.

For any compound, the therapeutically effective amount can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models, usually rats, mice, rabbits, dogs, or pigs. The animalmodel may also be used to determine the appropriate concentration rangeand route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.Therapeutic/prophylactic efficacy and toxicity may be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., ED₅₀ (the dose therapeutically effective in 50% of thepopulation) and LD₅₀ (the dose lethal to 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex, and it can be expressed as the ratio, LD₅₀/ED₅₀. Pharmaceuticalcompositions that exhibit large therapeutic indices are preferred. Thedosage may vary within this range depending upon the dosage formemployed, sensitivity of the patient, and the route of administration.

Dosage and administration are adjusted to provide sufficient levels ofthe active agent(s) or to maintain the desired effect. Factors which maybe taken into account include the severity of the disease state, generalhealth of the subject, age, weight, and gender of the subject, diet,time and frequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

The pharmaceutical compositions containing active compounds disclosedherein may be manufactured in a manner that is generally known, e.g., bymeans of conventional mixing, dissolving, granulating, dragee-making,levigating, emulsifying, encapsulating, entrapping, or lyophilizingprocesses. Pharmaceutical compositions may be formulated in aconventional manner using one or more pharmaceutically acceptablecarriers comprising excipients and/or auxiliaries that facilitateprocessing of the active compounds into preparations that can be usedpharmaceutically. Of course, the appropriate formulation is dependentupon the route of administration chosen.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as mannitol and sorbitol, and sodium chloridein the composition. Prolonged absorption of the injectable compositionscan be brought about by including in the composition an agent whichdelays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof

Oral compositions generally include an inert diluent or an ediblepharmaceutically acceptable carrier. They can be enclosed in gelatincapsules or compressed into tablets. For the purpose of oral therapeuticadministration, the active compound can be incorporated with excipientsand used in the form of tablets, troches, or capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash,wherein the compound in the fluid carrier is applied orally and swishedand expectorated or swallowed. Pharmaceutically compatible bindingagents, and/or adjuvant materials can be included as part of thecomposition. The tablets, pills, capsules, troches and the like cancontain any of the following ingredients, or compounds of a similarnature: a binder such as microcrystalline cellulose, gum tragacanth orgelatin; an excipient such as starch or lactose, a disintegrating agentsuch as alginic acid, Primogel, or corn starch; a lubricant such asmagnesium stearate or Sterotes; a glidant such as colloidal silicondioxide; a sweetening agent such as sucrose or saccharin; or a flavoringagent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser, whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The active compounds can be prepared with pharmaceutically acceptablecarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms disclosed herein are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved.

In therapeutic applications, the dosages of the EZH2 inhibitor compoundsdescribed herein, or the pharmaceutical compositions used in accordancewith the invention vary depending on the agent, the age, weight, andclinical condition of the recipient patient, and the experience andjudgment of the clinician or practitioner administering the therapy,among other factors affecting the selected dosage. Generally, the doseshould be sufficient to result in slowing, and preferably regressing,the growth of the tumors and also preferably causing complete regressionof the cancer. Dosages can range from about 0.01 mg/kg per day to about5000 mg/kg per day. In preferred aspects, dosages can range from about 1mg/kg per day to about 1000 mg/kg per day. In an aspect, the dose willbe in the range of about 0.1 mg/day to about 50 g/day; about 0.1 mg/dayto about 25 g/day; about 0.1 mg/day to about 10 g/day; about 0.1 mg toabout 3 g/day; or about 0.1 mg to about 1 g/day, in single, divided, orcontinuous doses (which dose may be adjusted for the patient's weight inkg, body surface area in m², and age in years). An effective amount of apharmaceutical agent is that which provides an objectively identifiableimprovement as noted by the clinician or other qualified observer. Forexample, regression of a tumor in a patient may be measured withreference to the diameter of a tumor. Decrease in the diameter of atumor indicates regression. Regression is also indicated by failure oftumors to reoccur after treatment has stopped. As used herein, the term“dosage effective manner” refers to amount of an active compound toproduce the desired biological effect in a subject or cell.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

The composition disclosed herein is capable of further forming salts.The composition disclosed herein is capable of forming more than onesalt per molecule, e.g., mono-, di-, tri-. All of these forms are alsocontemplated within the scope of the claimed invention.

As used herein, “pharmaceutically acceptable salts” refer to derivativesof the compounds disclosed herein wherein the parent compound ismodified by making acid or base salts thereof. Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines, alkalior organic salts of acidic residues such as carboxylic acids, and thelike. The pharmaceutically acceptable salts include the conventionalnon-toxic salts or the quaternary ammonium salts of the parent compoundformed, for example, from non-toxic inorganic or organic acids. Forexample, such conventional non-toxic salts include, but are not limitedto, those derived from inorganic and organic acids selected from2-acetoxybenzoic, 2-hydroxyethane sulfonic, acetic, ascorbic, benzenesulfonic, benzoic, bicarbonic, carbonic, citric, edetic, ethanedisulfonic, 1,2-ethane sulfonic, fumaric, glucoheptonic, gluconic,glutamic, glycolic, glycollyarsanilic, hexylresorcinic, hydrabamic,hydrobromic, hydrochloric, hydroiodic, hydroxymaleic, hydroxynaphthoic,isethionic, lactic, lactobionic, lauryl sulfonic, maleic, malic,mandelic, methane sulfonic, napsylic, nitric, oxalic, pamoic,pantothenic, phenylacetic, phosphoric, polygalacturonic, propionic,salicyclic, stearic, subacetic, succinic, sulfamic, sulfanilic,sulfuric, tannic, tartaric, toluene sulfonic, and the commonly occurringamine acids, e.g., glycine, alanine, phenylalanine, arginine, etc.

Other examples of pharmaceutically acceptable salts include hexanoicacid, cyclopentane propionic acid, pyruvic acid, malonic acid,3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, 4-chlorobenzenesulfonicacid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid,camphorsulfonic acid, 4-methylbicyclo-[2.2.2]-oct-2-ene-1-carboxylicacid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylaceticacid, muconic acid, and the like. The present invention also encompassessalts formed when an acidic proton present in the parent compound eitheris replaced by a metal ion, e.g., an alkali metal ion, an alkaline earthion, or an aluminum ion; or coordinates with an organic base such asethanolamine, diethanolamine, triethanolamine, tromethamine,N-methylglucamine, and the like.

It should be understood that all references to pharmaceuticallyacceptable salts include solvent addition forms (solvates), of the samesalt.

The composition disclosed herein may also be prepared as esters, forexample, pharmaceutically acceptable esters. For example, a carboxylicacid function group in a compound can be converted to its correspondingester, e.g., a methyl, ethyl or other ester. Also, an alcohol group in acompound can be converted to its corresponding ester, e.g., acetate,propionate or other ester.

The composition, or pharmaceutically acceptable salts orsolvatesthereof, are administered orally, nasally, transdermally,pulmonary, inhalationally, buccally, sublingually, intraperintoneally,subcutaneously, intramuscularly, intravenously, rectally,intrapleurally, intrathecally and parenterally. In one embodiment, thecompound is administered orally. One skilled in the art will recognizethe advantages of certain routes of administration.

The dosage regimen utilizing the compounds is selected in accordancewith a variety of factors including type, species, age, weight, sex andmedical condition of the patient; the severity of the condition to betreated; the route of administration; the renal and hepatic function ofthe patient; and the particular compound or salt thereof employed. Anordinarily skilled physician or veterinarian can readily determine andprescribe the effective amount of the drug required to prevent, counter,or arrest the progress of the condition.

Techniques for formulation and administration of the disclosed compoundscan be found in Remington: the Science and Practice of Pharmacy, 19thedition, Mack Publishing Co., Easton, Pa. (1995). In an embodiment, thecompounds described herein, and the pharmaceutically acceptable saltsthereof, are used in pharmaceutical preparations in combination with apharmaceutically acceptable carrier or diluent. Suitablepharmaceutically acceptable carriers include inert solid fillers ordiluents and sterile aqueous or organic solutions. The compounds will bepresent in such pharmaceutical compositions in amounts sufficient toprovide the desired dosage amount in the range described herein.

All percentages and ratios used herein, unless otherwise indicated, areby weight. Other features and advantages of the present disclosure areapparent from the different examples. The provided examples illustratedifferent components and methodology useful in practicing the presentinvention. The examples do not limit the claimed invention. Based on thepresent disclosure the skilled artisan can identify and employ othercomponents and methodology useful for practicing the present invention.

As used herein, a “subject in need thereof” is a subject having a NHL ora subject having an increased risk of developing such disorder relativeto the population at large. A subject in need thereof can have aprecancerous condition. A “subject” includes a mammal. The mammal can bee.g., any mammal, e.g., a human, primate, bird, mouse, rat, fowl, dog,cat, cow, horse, goat, camel, sheep or a pig. Preferably, the mammal isa human.

The subject includes any human subject who has been diagnosed with, hassymptoms of, or is at risk of developing NHL including DLBCL, GCB DLBCL,non-germinal center DLBCL including ABC DLBCL, FL, PMBCL, and MZL. Thesubject disclosed herein includes any human subject expressing a mutantEZH2 or WT EZH2 or has a mutation in the EZH2 gene or has a wild-typeEZH2 gene. For example, a mutant EZH2 comprises one or more mutations,wherein the mutation is a substitution, a point mutation, a nonsensemutation, a missense mutation, a deletion, or an insertion or any otherEZH2 mutation described herein.

A subject in need thereof may have refractory or resistant cancer.“Refractory or resistant cancer” means cancer that does not respond totreatment. The cancer may be resistant at the beginning of treatment orit may become resistant during treatment. In some embodiments, thesubject in need thereof has cancer recurrence following remission onmost recent therapy. In some embodiments, the subject in need thereofreceived and failed all known effective therapies for cancer treatment.In some embodiments, the subject in need thereof received at least oneprior therapy. In certain embodiments the prior therapy is monotherapy.In certain embodiments the prior therapy is combination therapy.

In some embodiments, a subject in need thereof may have a secondarycancer as a result of a previous therapy. “Secondary cancer” meanscancer that arises due to or as a result from previous carcinogenictherapies, such as chemotherapy.

The subject may also exhibit resistance to EZH2 histonemethyltransferase inhibitors or any other therapeutic agent.

The invention also features a method of selecting a therapy for asubject having a lymphoma including DLBCL, GCB DLBCL, non-germinalcenter DLBCL, ABC DLBCL, FL, PMBCL, and MZL.

The method includes the steps of: detecting the presence or absence ofone or more EZH2 mutations described herein in a sample from thesubject; and selecting, based on the presence or absence of the one ormore EZH2 mutations, a therapy for treating the lymphoma. In oneembodiment, the therapy includes administering to the subject atherapeutically effective amount of an EZH2 inhibitor described herein.An EZH2 mutation or absence thereof can be detected using any suitablemethod known in the art.

The methods and uses described herein may include steps of detecting thepresence or absence of one or more EZH2 mutations described herein in asample from a subject in need thereof prior to and/or after theadministration of a composition disclosed herein (e.g., a compositioncomprising a compound disclosed herein or pharmaceutically acceptablesalts thereof, alone or in combination with one or more secondtherapeutic agents) to the subject.

The present invention provides personalized medicine, treatment and/orcancer management for a subject having or at risk of having a germinalcenter-derived lymphoma, by genetic screening of one or more EZH2mutations described herein in the subject. For example, the presentinvention provides methods for treating or alleviating a symptom of agerminal center-derived lymphoma in a subject in need thereof bydetermining responsiveness of the subject to a therapy and when thesubject is responsive to the therapy, administering to the subject acomposition disclosed herein. Once the responsiveness of a subject isdetermined, a therapeutically effective amount of a composition, forexample, a composition comprising a compound disclosed herein orpharmaceutically acceptable salts thereof, alone or in combination withone or more second therapeutic agents, can be administered. Thetherapeutically effective amount of a composition can be determined byone of ordinary skill in the art.

As used herein, the term “responsiveness” is interchangeable with terms“responsive”, “sensitive”, and “sensitivity”, and it is meant that asubject is showing therapeutic responses when administered a compositiondisclosed herein, e.g., tumor cells or tumor tissues of the subjectundergo apoptosis and/or necrosis, and/or display reduced growing,dividing, or proliferation. This term is also meant that a subject willor has a higher probability, relative to the population at large, ofshowing therapeutic responses when administered a composition disclosedherein, e.g., tumor cells or tumor tissues of the subject undergoapoptosis and/or necrosis, and/or display reduced growing, dividing, orproliferation.

By “sample” it means any biological sample derived from the subject,includes but is not limited to, cells, tissues samples, body fluids(including, but not limited to, mucus, blood, plasma, serum, urine,saliva, and semen), tumor cells, and tumor tissues. Preferably, thesample is selected from bone marrow, peripheral blood cells, blood,plasma and serum. Samples can be provided by the subject under treatmentor testing. Alternatively samples can be obtained by the physicianaccording to routine practice in the art.

As used herein, “candidate compound” refers to a compound disclosedherein, or a pharmaceutically acceptable salt or solvate thereof, thathas been or will be tested in one or more in vitro or in vivo biologicalassays, in order to determine if that compound is likely to elicit adesired biological or medical response in a cell, tissue, system, animalor human that is being sought by a researcher or clinician. A candidatecompound is a compound disclosed herein, or a pharmaceuticallyacceptable salt or solvate thereof. The biological or medical responsecan be the treatment of cancer. The biological or medical response canbe treatment or prevention of a cell proliferative disorder. In vitro orin vivo biological assays can include, but are not limited to, enzymaticactivity assays, electrophoretic mobility shift assays, reporter geneassays, in vitro cell viability assays, and the assays described herein.

As used herein, “treating” or “treat” describes the management and careof a patient for the purpose of combating a disease, condition, ordisorder and includes the administration of a compound disclosed herein,or a pharmaceutically acceptable salt or solvate thereof, to alleviatethe symptoms or complications of a disease, condition or disorder, or toeliminate the disease, condition or disorder.

A composition disclosed herein, or a pharmaceutically acceptable salt orsolvate thereof, can also be used to prevent a disease, condition ordisorder. As used herein, “preventing” or “prevent” describes reducingor eliminating the onset of the symptoms or complications of thedisease, condition or disorder.

As used herein, the term “alleviate” is meant to describe a process bywhich the severity of a sign or symptom of a disorder is decreased.Importantly, a sign or symptom can be alleviated without beingeliminated. In a preferred embodiment, the administration ofpharmaceutical compositions disclosed herein leads to the elimination ofa sign or symptom, however, elimination is not required. Effectivedosages are expected to decrease the severity of a sign or symptom. Forinstance, a sign or symptom of a disorder such as cancer, which canoccur in multiple locations, is alleviated if the severity of the canceris decreased within at least one of multiple locations.

As used herein, the term “severity” is meant to describe the potentialof cancer to transform from a precancerous, or benign, state into amalignant state. Alternatively, or in addition, severity is meant todescribe a cancer stage, for example, according to the TNM system(accepted by the International Union Against Cancer (UICC) and theAmerican Joint Committee on Cancer (AJCC)) or by other art-recognizedmethods. Cancer stage refers to the extent or severity of the cancer,based on factors such as the location of the primary tumor, tumor size,number of tumors, and lymph node involvement (spread of cancer intolymph nodes). Alternatively, or in addition, severity is meant todescribe the tumor grade by art-recognized methods (see, National CancerInstitute, www.cancer.gov). Tumor grade is a system used to classifycancer cells in terms of how abnormal they look under a microscope andhow quickly the tumor is likely to grow and spread. Many factors areconsidered when determining tumor grade, including the structure andgrowth pattern of the cells. The specific factors used to determinetumor grade vary with each type of cancer. Severity also describes ahistologic grade, also called differentiation, which refers to how muchthe tumor cells resemble normal cells of the same tissue type (see,National Cancer Institute, www.cancer.gov). Furthermore, severitydescribes a nuclear grade, which refers to the size and shape of thenucleus in tumor cells and the percentage of tumor cells that aredividing (see, National Cancer Institute, www. cancer. gov).

In another aspect, severity describes the degree to which a tumor hassecreted growth factors, degraded the extracellular matrix, becomevascularized, lost adhesion to juxtaposed tissues, or metastasized.Moreover, severity describes the number of locations to which a primarytumor has metastasized. Finally, severity includes the difficulty oftreating tumors of varying types and locations. For example, inoperabletumors, those cancers which have greater access to multiple body systems(hematological and immunological tumors), and those which are the mostresistant to traditional treatments are considered most severe. In thesesituations, prolonging the life expectancy of the subject and/orreducing pain, decreasing the proportion of cancerous cells orrestricting cells to one system, and improving cancer stage/tumorgrade/histological grade/nuclear grade are considered alleviating a signor symptom of the cancer.

As used herein the term “symptom” is defined as an indication ofdisease, illness, injury, or that something is not right in the body.Symptoms are felt or noticed by the individual experiencing the symptom,but may not easily be noticed by others. Others are defined asnon-health-care professionals.

As used herein the term “sign” is also defined as an indication thatsomething is not right in the body. But signs are defined as things thatcan be seen by a doctor, nurse, or other health care professional.

Cancer is a group of diseases that may cause almost any sign or symptom.The signs and symptoms will depend on where the cancer is, the size ofthe cancer, and how much it affects the nearby organs or structures. Ifa cancer spreads (metastasizes), then symptoms may appear in differentparts of the body.

Treating cancer can result in a reduction in size of a tumor. Areduction in size of a tumor may also be referred to as “tumorregression”. Preferably, after treatment, tumor size is reduced by 5% orgreater relative to its size prior to treatment; more preferably, tumorsize is reduced by 10% or greater; more preferably, reduced by 20% orgreater; more preferably, reduced by 30% or greater; more preferably,reduced by 40% or greater; even more preferably, reduced by 50% orgreater; and most preferably, reduced by greater than 75% or greater.Size of a tumor may be measured by any reproducible means ofmeasurement. The size of a tumor may be measured as a diameter of thetumor.

Treating cancer can result in a reduction in tumor volume. Preferably,after treatment, tumor volume is reduced by 5% or greater relative toits size prior to treatment; more preferably, tumor volume is reduced by10% or greater; more preferably, reduced by 20% or greater; morepreferably, reduced by 30% or greater; more preferably, reduced by 40%or greater; even more preferably, reduced by 50% or greater; and mostpreferably, reduced by greater than 75% or greater. Tumor volume may bemeasured by any reproducible means of measurement.

Treating cancer results in a decrease in number of tumors. Preferably,after treatment, tumor number is reduced by 5% or greater relative tonumber prior to treatment; more preferably, tumor number is reduced by10% or greater; more preferably, reduced by 20% or greater; morepreferably, reduced by 30% or greater; more preferably, reduced by 40%or greater; even more preferably, reduced by 50% or greater; and mostpreferably, reduced by greater than 75%. Number of tumors may bemeasured by any reproducible means of measurement. The number of tumorsmay be measured by counting tumors visible to the naked eye or at aspecified magnification. Preferably, the specified magnification is 2×,3×, 4×, 5×, 10×, or 50×.

Treating cancer can result in a decrease in number of metastatic lesionsin other tissues or organs distant from the primary tumor site.Preferably, after treatment, the number of metastatic lesions is reducedby 5% or greater relative to number prior to treatment; more preferably,the number of metastatic lesions is reduced by 10% or greater; morepreferably, reduced by 20% or greater; more preferably, reduced by 30%or greater; more preferably, reduced by 40% or greater; even morepreferably, reduced by 50% or greater; and most preferably, reduced bygreater than 75%. The number of metastatic lesions may be measured byany reproducible means of measurement. The number of metastatic lesionsmay be measured by counting metastatic lesions visible to the naked eyeor at a specified magnification. Preferably, the specified magnificationis 2×, 3×, 4×, 5×, 10×, or 50×.

Treating cancer can result in an increase in average survival time of apopulation of treated subjects in comparison to a population receivingcarrier alone. Preferably, the average survival time is increased bymore than 30 days; more preferably, by more than 60 days; morepreferably, by more than 90 days; and most preferably, by more than 120days. An increase in average survival time of a population may bemeasured by any reproducible means. An increase in average survival timeof a population may be measured, for example, by calculating for apopulation the average length of survival following initiation oftreatment with an active compound. An increase in average survival timeof a population may also be measured, for example, by calculating for apopulation the average length of survival following completion of afirst round of treatment with an active compound.

Treating cancer can result in an increase in average survival time of apopulation of treated subjects in comparison to a population ofuntreated subjects. Preferably, the average survival time is increasedby more than 30 days; more preferably, by more than 60 days; morepreferably, by more than 90 days; and most preferably, by more than 120days. An increase in average survival time of a population may bemeasured by any reproducible means. An increase in average survival timeof a population may be measured, for example, by calculating for apopulation the average length of survival following initiation oftreatment with an active compound. An increase in average survival timeof a population may also be measured, for example, by calculating for apopulation the average length of survival following completion of afirst round of treatment with an active compound.

Treating cancer can result in increase in average survival time of apopulation of treated subjects in comparison to a population receivingmonotherapy with a drug that is not a compound disclosed herein, or apharmaceutically acceptable salt, solvate, analog or derivative thereof.Preferably, the average survival time is increased by more than 30 days;more preferably, by more than 60 days; more preferably, by more than 90days; and most preferably, by more than 120 days. An increase in averagesurvival time of a population may be measured by any reproducible means.An increase in average survival time of a population may be measured,for example, by calculating for a population the average length ofsurvival following initiation of treatment with an active compound. Anincrease in average survival time of a population may also be measured,for example, by calculating for a population the average length ofsurvival following completion of a first round of treatment with anactive compound.

Treating cancer can result in a decrease in the mortality rate of apopulation of treated subjects in comparison to a population receivingcarrier alone. Treating cancer can result in a decrease in the mortalityrate of a population of treated subjects in comparison to an untreatedpopulation. Treating cancer can result in a decrease in the mortalityrate of a population of treated subjects in comparison to a populationreceiving monotherapy with a drug that is not a compound disclosedherein, or a pharmaceutically acceptable salt, solvate, analog orderivative thereof. Preferably, the mortality rate is decreased by morethan 2%; more preferably, by more than 5%; more preferably, by more than10%; and most preferably, by more than 25%. A decrease in the mortalityrate of a population of treated subjects may be measured by anyreproducible means. A decrease in the mortality rate of a population maybe measured, for example, by calculating for a population the averagenumber of disease-related deaths per unit time following initiation oftreatment with an active compound. A decrease in the mortality rate of apopulation may also be measured, for example, by calculating for apopulation the average number of disease-related deaths per unit timefollowing completion of a first round of treatment with an activecompound.

Treating cancer can result in a decrease in tumor growth rate.Preferably, after treatment, tumor growth rate is reduced by at least 5%relative to number prior to treatment; more preferably, tumor growthrate is reduced by at least 10%; more preferably, reduced by at least20%; more preferably, reduced by at least 30%; more preferably, reducedby at least 40%; more preferably, reduced by at least 50%; even morepreferably, reduced by at least 50%; and most preferably, reduced by atleast 75%. Tumor growth rate may be measured by any reproducible meansof measurement. Tumor growth rate can be measured according to a changein tumor diameter per unit time.

Treating cancer can result in a decrease in tumor regrowth. Preferably,after treatment, tumor regrowth is less than 5%; more preferably, tumorregrowth is less than 10%; more preferably, less than 20%; morepreferably, less than 30%; more preferably, less than 40%; morepreferably, less than 50%; even more preferably, less than 50%; and mostpreferably, less than 75%. Tumor regrowth may be measured by anyreproducible means of measurement. Tumor regrowth is measured, forexample, by measuring an increase in the diameter of a tumor after aprior tumor shrinkage that followed treatment. A decrease in tumorregrowth is indicated by failure of tumors to reoccur after treatmenthas stopped.

Treating or preventing a cell proliferative disorder can result in areduction in the rate of cellular proliferation. Preferably, aftertreatment, the rate of cellular proliferation is reduced by at least 5%;more preferably, by at least 10%; more preferably, by at least 20%; morepreferably, by at least 30%; more preferably, by at least 40%; morepreferably, by at least 50%; even more preferably, by at least 50%; andmost preferably, by at least 75%. The rate of cellular proliferation maybe measured by any reproducible means of measurement. The rate ofcellular proliferation is measured, for example, by measuring the numberof dividing cells in a tissue sample per unit time.

Treating or preventing a cell proliferative disorder can result in areduction in the proportion of proliferating cells. Preferably, aftertreatment, the proportion of proliferating cells is reduced by at least5%; more preferably, by at least 10%; more preferably, by at least 20%;more preferably, by at least 30%; more preferably, by at least 40%; morepreferably, by at least 50%; even more preferably, by at least 50%; andmost preferably, by at least 75%. The proportion of proliferating cellsmay be measured by any reproducible means of measurement. Preferably,the proportion of proliferating cells is measured, for example, byquantifying the number of dividing cells relative to the number ofnondividing cells in a tissue sample. The proportion of proliferatingcells can be equivalent to the mitotic index.

Treating or preventing a cell proliferative disorder can result in adecrease in size of an area or zone of cellular proliferation.Preferably, after treatment, size of an area or zone of cellularproliferation is reduced by at least 5% relative to its size prior totreatment; more preferably, reduced by at least 10%; more preferably,reduced by at least 20%; more preferably, reduced by at least 30%; morepreferably, reduced by at least 40%; more preferably, reduced by atleast 50%; even more preferably, reduced by at least 50%; and mostpreferably, reduced by at least 75%. Size of an area or zone of cellularproliferation may be measured by any reproducible means of measurement.The size of an area or zone of cellular proliferation may be measured asa diameter or width of an area or zone of cellular proliferation.

Treating or preventing a cell proliferative disorder can result in adecrease in the number or proportion of cells having an abnormalappearance or morphology. Preferably, after treatment, the number ofcells having an abnormal morphology is reduced by at least 5% relativeto its size prior to treatment; more preferably, reduced by at least10%; more preferably, reduced by at least 20%; more preferably, reducedby at least 30%; more preferably, reduced by at least 40%; morepreferably, reduced by at least 50%; even more preferably, reduced by atleast 50%; and most preferably, reduced by at least 75%. An abnormalcellular appearance or morphology may be measured by any reproduciblemeans of measurement. An abnormal cellular morphology can be measured bymicroscopy, e.g., using an inverted tissue culture microscope. Anabnormal cellular morphology can take the form of nuclear pleiomorphism.

As used herein, the term “selectively” means tending to occur at ahigher frequency in one population than in another population. Thecompared populations can be cell populations. Preferably, a compounddisclosed herein, or a pharmaceutically acceptable salt or solvatethereof, acts selectively on a cancer or precancerous cell but not on anormal cell. Preferably, a compound disclosed herein, or apharmaceutically acceptable salt or solvate thereof, acts selectively tomodulate one molecular target (e.g., a target protein methyltransferase)but does not significantly modulate another molecular target (e.g., anon-target protein methyltransferase). The invention also provides amethod for selectively inhibiting the activity of an enzyme, such as aprotein methyltransferase. Preferably, an event occurs selectively inpopulation A relative to population B if it occurs greater than twotimes more frequently in population A as compared to population B. Anevent occurs selectively if it occurs greater than five times morefrequently in population A. An event occurs selectively if it occursgreater than ten times more frequently in population A; more preferably,greater than fifty times; even more preferably, greater than 100 times;and most preferably, greater than 1000 times more frequently inpopulation A as compared to population B. For example, cell death wouldbe said to occur selectively in cancer cells if it occurred greater thantwice as frequently in cancer cells as compared to normal cells.

A composition disclosed herein, e.g., a composition comprising anycompound disclosed herein or pharmaceutically acceptable salt thereof,can modulate the activity of a molecular target (e.g., a target proteinmethyltransferase). Modulating refers to stimulating or inhibiting anactivity of a molecular target. Preferably, a compound disclosed herein,or a pharmaceutically acceptable salt or solvate thereof, modulates theactivity of a molecular target if it stimulates or inhibits the activityof the molecular target by at least 2-fold relative to the activity ofthe molecular target under the same conditions but lacking only thepresence of said compound. More preferably, a compound disclosed herein,or a pharmaceutically acceptable salt or solvate thereof, modulates theactivity of a molecular target if it stimulates or inhibits the activityof the molecular target by at least 5-fold, at least 10-fold, at least20-fold, at least 50-fold, at least 100-fold relative to the activity ofthe molecular target under the same conditions but lacking only thepresence of said compound. The activity of a molecular target may bemeasured by any reproducible means. The activity of a molecular targetmay be measured in vitro or in vivo. For example, the activity of amolecular target may be measured in vitro by an enzymatic activity assayor a DNA binding assay, or the activity of a molecular target may bemeasured in vivo by assaying for expression of a reporter gene.

A composition disclosed herein does not significantly modulate theactivity of a molecular target if the addition of the compound does notstimulate or inhibit the activity of the molecular target by greaterthan 10% relative to the activity of the molecular target under the sameconditions but lacking only the presence of said compound.

As used herein, the term “isozyme selective” means preferentialinhibition or stimulation of a first isoform of an enzyme in comparisonto a second isoform of an enzyme (e.g., preferential inhibition orstimulation of a protein methyltransferase isozyme alpha in comparisonto a protein methyltransferase isozyme beta). Preferably, a compounddisclosed herein, or a pharmaceutically acceptable salt or solvatethereof, demonstrates a minimum of a fourfold differential, preferably atenfold differential, more preferably a fifty fold differential, in thedosage required to achieve a biological effect. Preferably, a compounddisclosed herein, or a pharmaceutically acceptable salt or solvatethereof, demonstrates this differential across the range of inhibition,and the differential is exemplified at the IC_(50,) i.e., a 50%inhibition, for a molecular target of interest.

Administering a composition disclosed herein to a cell or a subject inneed thereof can result in modulation (i.e., stimulation or inhibition)of an activity of a protein methyltransferase of interest.

Administering a compound disclosed herein, e.g., a compositioncomprising any compound disclosed herein or pharmaceutically acceptablesalt thereof, and one or more other therapeutic agents, such asprednisone, to a cell or a subject in need thereof results in modulation(i.e., stimulation or inhibition) of an activity of an intracellulartarget (e.g., substrate). Several intracellular targets can be modulatedwith the compounds disclosed herein, including, but not limited to,protein methyltrasferase.

Activating refers to placing a composition of matter (e.g., protein ornucleic acid) in a state suitable for carrying out a desired biologicalfunction. A composition of matter capable of being activated also has anunactivated state. An activated composition of matter may have aninhibitory or stimulatory biological function, or both.

Elevation refers to an increase in a desired biological activity of acomposition of matter (e.g., a protein or a nucleic acid). Elevation mayoccur through an increase in concentration of a composition of matter.

Treating cancer or a cell proliferative disorder can result in celldeath, and preferably, cell death results in a decrease of at least 10%in number of cells in a population. More preferably, cell death means adecrease of at least 20%; more preferably, a decrease of at least 30%;more preferably, a decrease of at least 40%; more preferably, a decreaseof at least 50%; most preferably, a decrease of at least 75%. Number ofcells in a population may be measured by any reproducible means. Anumber of cells in a population can be measured by fluorescenceactivated cell sorting (FACS), immunofluorescence microscopy and lightmicroscopy. Methods of measuring cell death are as shown in Li et al.,Proc Natl Acad Sci USA. 100(5): 2674-8, 2003. In an aspect, cell deathoccurs by apoptosis.

Preferably, an effective amount of a composition disclosed herein, or apharmaceutically acceptable salt or solvate thereof, is notsignificantly cytotoxic to normal cells. A therapeutically effectiveamount of a compound is not significantly cytotoxic to normal cells ifadministration of the compound in a therapeutically effective amountdoes not induce cell death in greater than 10% of normal cells. Atherapeutically effective amount of a compound does not significantlyaffect the viability of normal cells if administration of the compoundin a therapeutically effective amount does not induce cell death ingreater than 10% of normal cells. In an aspect, cell death occurs byapoptosis.

Contacting a cell with a composition disclosed herein, or apharmaceutically acceptable salt or solvate thereof, can induce oractivate cell death selectively in cancer cells. Administering to asubject in need thereof a compound disclosed herein, or apharmaceutically acceptable salt or solvate thereof, can induce oractivate cell death selectively in cancer cells. Contacting a cell witha composition disclosed herein, or a pharmaceutically acceptable salt orsolvate thereof, can induce cell death selectively in one or more cellsaffected by a cell proliferative disorder. Preferably, administering toa subject in need thereof a composition disclosed herein, or apharmaceutically acceptable salt or solvate thereof, induces cell deathselectively in one or more cells affected by a cell proliferativedisorder.

The present disclosure relates to a method of treating or preventingcancer by administering a composition disclosed herein, or apharmaceutically acceptable salt or solvate thereof, to a subject inneed thereof, where administration of the composition disclosed herein,or a pharmaceutically acceptable salt or solvate thereof, results in oneor more of the following: prevention of cancer cell proliferation byaccumulation of cells in one or more phases of the cell cycle (e.g. G1,G1/S, G2/M), or induction of cell senescence, or promotion of tumor celldifferentiation; promotion of cell death in cancer cells viacytotoxicity, necrosis or apoptosis, without a significant amount ofcell death in normal cells, antitumor activity in animals with atherapeutic index of at least 2. As used herein, “therapeutic index” isthe maximum tolerated dose divided by the efficacious dose.

One skilled in the art may refer to general reference texts for detaileddescriptions of known techniques discussed herein or equivalenttechniques. These texts include Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Inc. (2005); Sambrook et al.,Molecular Cloning, A Laboratory Manual (3rd edition), Cold Spring HarborPress, Cold Spring Harbor, N.Y. (2000); Coligan et al., CurrentProtocols in Immunology, John Wiley & Sons, N.Y.; Enna et al., CurrentProtocols in Pharmacology, John Wiley & Sons, N.Y.; Fingl et al., ThePharmacological Basis of Therapeutics (1975), Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., 18t^(h) edition (1990).These texts can, of course, also be referred to in making or using anaspect of the invention.

EXAMPLE 1 EPZ-6438 Clinical Study

The phase 1 trial enrolled patients with relapsed/refractory solidtumors and B cell lymphoma. The study employed a standard 3+3 doseescalation design with two planned dose expansion cohorts and clinicalpharmacology sub-studies. The primary endpoint was determination of arecommended phase 2 dose or MTD with standard secondary endpoints.

The patients enrolled included 19 patients with NHL of which 13 patientshave DLBCL. Cell-of origin testing was intended for all NHL patients,however, 3 DLBCL patients had insufficient tissue to permitdetermination of germinal center vs. non-germinal center status. EZH2mutation testing was centrally performed for 14 NHL patients by thecobas® test (Roche). One lymphoma patient whose tumor has been treatedto date carries an EZH2 mutation. For the solid tumor patients,attention has been given to recruiting patients with INI1-deficienttumors due to the oncogenic role of EZH2 in these tumors.

NHL patients on study were heavily pre-treated with 85% having receivedthree or more prior systemic therapies and nearly half receiving four ormore prior regimens. 37% were refractory to their most recent priorregimen and five patients had a prior transplant.

The pharmacokinetics of EPZ-6438 are characterized by rapid absorptionand a terminal half-life of 3 to 5 hours (FIG. 4). The drug displaysdose-proportional linear PK at steady state throughout the entire dosingrange. While a decrease in AUC between the first dose and day 15 wasobserved, there was no further reduction in systemic exposure beyondthat time, as evidenced by pre-dose Ctrou_(g)h levels in the rightpanel.

FIG. 5 shows the pre- and post-dose skin biopsies were collected toassess pharmacodynamics in a surrogate tissue through the measurement oftri-methyl H3K27 levels by immunohistochemistry. It was previously shownthat dose-dependent reduction of tri-methyl H3K27 levels across the fullthickness of skin as demonstrated in the top right panel. With furtherrefined quantitation by using image analysis to assess H3K27 signal indifferent layers of the skin; a much greater reduction of tri-methylH3K27 signal was observed in the spinosum layer versus the basal layerwhich does not change appreciably.

These differences in pharmacodynamic response between the differentlayers of skin highlight the potential for variability in the kineticsfor tri-methyl H3K27 turnover, even in cells of the same tissue.

EPZ-6438 is well-tolerated with the most common adverse events beingasthenia, anorexia, anemia, dyspnea and nausea across the entirepopulation (FIG. 6).

-   Grade 3 or greater adverse events were observed in fewer than a    third of patients.-   Grade 3 or greater treatment-related adverse events were observed in    only 5 patients.-   The only DLT observed was thrombocytopenia which occurred at 1600    mg.-   One patient required a dose reduction for thrombocytopenia. One    patient discontinued drug for grade 4 neutropenia. Both of these    patients were treated in the 800 mg expansion cohort.-   Seven patients had dose interruptions. Of these, 6 were from a    reversible toxicity and resumed study agent at prior dose without    further issue.

FIG. 7 shows that of 15 evaluable NHL patients, 9 have had an objectiveresponse.

-   In DLBCL, objective responses were seen in 5 of 9 patients. Of    these, one patient remained on study at over 18 months and an    additional patient with an EZH2 mutation remained on study at 6    months.-   In follicular lymphoma, 3 of 5 patients achieved objective responses    with two patients on study at 12 months.-   One patient with marginal zone nodular lymphoma remained on study    with a gradually improving partial response approaching one year on    therapy.

One feature of EPZ-6438's anti-tumor activity in NHL is a gradual, butprolonged reduction in tumor mass. This results in an evolution ofobjective response which can occur through 10 months on study. Patientsmay have a prolonged period of SD with gradual tumor shrinkage beforebecoming a PR. And the same may be seen before a PR becomes a CR. Thispattern of response was observed across all subtypes of NHL studied todate.

FIG. 9 shows a 23 year old male with primary mediastinal B-cell lymphomawho became a CR by week 40 with a negative PET, despite being refractoryto multiple intensive rituximab+chemotherapy regimens. He remained in CRat week 78.

FIG. 10 shows a male with multiply refractory follicular lymphoma asanother example of evolution of response. His pen-orbital tumor reachedcriteria for PR at week 16 and then CR by week 32. He remained in CR atweek 60.

FIG. 11 shows a patient with a tumor bearing an EZH2 mutation. She hasan aggressive DLBCL and was very heavily pre-treated with six priorregimens and without an objective response for the last 3 years. Herscans revealed a dramatic response to tazemetostat with a 52% reductionof a very large abdominal mass by week 16. She remained in PR through 24weeks on study.

All publications and patent documents cited herein are incorporatedherein by reference as if each such publication or document wasspecifically and individually indicated to be incorporated herein byreference. Citation of publications and patent documents is not intendedas an admission that any is pertinent prior art, nor does it constituteany admission as to the contents or date of the same. The inventionhaving now been described by way of written description, those of skillin the art will recognize that the invention can be practiced in avariety of embodiments and that the foregoing description and examplesbelow are for purposes of illustration and not limitation of the claimsthat follow.

The invention can be embodied in other specific forms without departingfrom the spirit or essential characteristics thereof The foregoingembodiments are therefore to be considered in all respects illustrativerather than limiting on the invention described herein. Scope of theinvention is thus indicated by the appended claims rather than by theforegoing description, and all changes that come within the meaning andrange of equivalency of the claims are intended to be embraced therein.

1. A method for treating non-Hodgkin's lymphoma (NHL) comprisingadministering a therapeutically effective amount of an EZH2 inhibitor toa subject in need thereof, wherein the NHL is selected from diffuselarge B-cell lymphoma (DLBCL), a germinal center-derived lymphoma, anon-germinal center-derived lymphoma, follicular lymphoma (FL), primarymediastinal large B-cell lymphoma (PMBCL), marginal zone lymphoma (MZL),Burkitt's lymphoma and other non-Hodgkin's lymphoma subtype.
 2. Themethod of claim 1, wherein the NHL is DLBCL.
 3. The method of claim 1,wherein the NHL is a germinal center-derived lymphoma.
 4. The method ofclaim 1, wherein the NHL is a non-germinal center-derived lymphoma. 5.The method of claim 1, wherein the NHL is follicular lymphoma.
 6. Themethod of claim 1, wherein the NHL is PMBCL.
 7. The method of claim 1,wherein the NHL is marginal zone lymphoma.
 8. The method of claim 1,wherein the NHL is Burkitt's lymphoma.
 9. The method of claim 1, whereinthe NHL is other Non-Hodgkin's lymphoma subtype.
 10. The method of claim1, wherein the NHL is an EZH2 wild type B-cell lymphoma.
 11. The methodof claim 1, wherein the NHL is an EZH2 mutant B-cell lymphoma.
 12. Themethod of claim 1, wherein the EZH2 inhibitor is administered orally.13. The method of claim 1, wherein the subject is a human being.
 14. Themethod of claim 13, wherein the EZH2 inhibitor is EPZ-6438 having thefollowing formula:

or a pharmaceutically acceptable salt thereof.
 15. The method of claim14, wherein the EZH2 inhibitor is administered to the subject at a doseof about 100 mg to about 3200 mg daily.
 16. The method of claim 14,wherein the EZH2 inhibitor is administered to the subject at a dose ofabout 100 mg BID to about 1600 mg BID.
 17. The method of claim 14,wherein the EZH2 inhibitor is administered to the subject at a dose ofabout 100 mg BID, 200 mg BID, 400 mg BID, 800 mg BID, or about 1600 mgBID.
 18. The method of claim 17 wherein the EZH2 inhibitor isadministered to the subject at a dose of 800 mg BID.
 19. The method ofclaim 13, wherein the EZH2 inhibitor is:

or a pharmaceutically acceptable salt thereof.
 20. The method of claim19, wherein the EZH2 inhibitor is:

or a pharmaceutically acceptable salt thereof.